The physiology of paracellular permeation of ions and solutes in the kidney is pivotally important but poorly understood. Claudins are the key components of the paracellular pathway. Defects in claudin function result in a broad range of renal diseases, including hypomagnesemia, hypercalciuria, hypochloremia and salt- sensitive hypertension. Human mutations in claudin-16 and claudin-19 are responsible for the hereditary renal disease FHHNC (Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis). We have found that the two claudins co-localize in the thick ascending limb (TAL), interact and form a cation-selective protein complex. We have developed claudin-16 knockdown (KD) and claudin-19 KD mouse lines. Claudin-16 KD animals show chronic renal wasting of magnesium and calcium, developing renal nephrocalcinosis comparable to that seen in human patients with FHHNC. This proposal will study the molecular interaction between claudin-16 and claudin-19, the size and charge selectivity of claudin-16 and claudin-19 channel pore and their collective effects on renal handling of Mg++. Specific Aim 1 intends to understand how claudin-16 and claudin-19 co-oligomerize and assemble into the tight junction strands. These studies will determine the stoichiometry of claudin-16/claudin- 19 oligomeric complex and identify critical intracellular step in claudin oligomerization using metabolic chase analyses. Specific Aim 2 intends to elucidate the biophysical properties of the claudin-16 and claudin-19 channels. These studies will measure the paracellular flux of a continuous series of PEG oligomers (of radius 2.8 - 7 ) to unveil the size selectivity of claudin-16 and claudin-19 channels. These studies will identify key loci of amino acids in claudin-16 and claudin-19 required for their charge selectivity. These studies will test two models of claudin-16 and claudin-19 channel structure and function: (1) claudin-16 and claudin-19 form two parallel homomeric channels each with its own physiologic signature; (2) claudin-16 and claudin-19 form a heteromeric channel with novel properties that require their synergy. Specific Aim 3 involves careful phenotypic analyses of claudin-16 KD mice, claudin-19 KD (KO) mice and claudin-16 KD + claudin-19 KD (KO) mice. These studies will analyze the renal clearance and transport functions in these mice, and record the electrophysiological properties of claudin-16 and claudin-19 channels in the TAL of these mice using single tubule perfusion techniques. These studies intend to test if the interaction between claudin-16 and claudin-19 is required for normal function of the TAL in vivo.